Two primary modes of chemical communication occur between neurons in the brain: fast synaptic transmission, such as that mediated by glutamate and GABA, which directly control the electrical activities of neurons, and slow synaptic transmission, such as that mediated by norepinephrine and dopamine, which regulate subcellular signaling events that cannot be measured directly from neuronal electrical activities. Slow synaptic transmission, which is also called neuromodulation, plays important modulatory roles in regulating excitability, synaptic plasticity and other aspects of neuronal function, and eventually imposes powerful control over the function of fast synaptic transmission. However, unlike fast synaptic transmission, which can be monitored directly via an increasing number of modern approaches such as multi-electrode recording, voltage imaging and calcium imaging methods, much less is known about the precise neuromodulatory events that occur in living animals because there has not been an established method to reliably record the relevant activities triggered by neuromodulation in individual neurons in vivo. To overcome this problem, we propose a novel approach for examining neuromodulatory activities with single-neuron resolution in vivo by imaging the activity of cyclic AMP (cAMP) and protein kinase A (PKA). The cAMP/PKA pathway is a common downstream signal transduction pathway for both dopamine and norepinephrine. Although genetically encoded cAMP/PKA sensors based on Frster resonance energy transfer (FRET) have been used for experiments in vitro, their application in vivo has been difficult due to lower signal-to-noise ratios under the more challenging in vivo imaging conditions. We propose a multipronged approach to eliminate several bottlenecks encountered with current FRET imaging approaches to maximize the signal-to-noise ratio. Our approach includes: 1) developing and improving cAMP/PKA sensors, 2) implementing a FRET imaging modality that is more effective than conventional FRET measures in light-scattering brain tissue, 3) correcting light aberrations associated with in vivo imaging conditions, and 4) developing novel mouse reagents for high-contrast, reproducible FRET imaging. We will validate the utility of this method for monitoring neuromodulatory activities by determining the spatiotemporal patterns of norepinephrine action in anesthetized mice using optogenetic approaches and in behaving mice using different stress stimulations. If successful, our efforts will provide a previously unattainable ability to conduct large- scale monitoring of neuromodulatory activities in the brain at the cellular and circuitry levels. This ability to quantitate neuromodulation will complement the measurements of fast synaptic transmission to enhance our understanding of brain function underlying animal behavior.